Defactinib inhibits FAK phosphorylation and regulates psoriasis via attenuating hyperproliferation of keratinocytes.

Excessive proliferation of keratinocytes is a crucial pathological risk feature of psoriasis. Focal adhesion kinase (FAK) is a non-receptor protein that primarily regulates cell proliferation and migration. However, the expression and regulatory mechanism of FAK in psoriasis remains unclear. This study aimed to investigate the regulation of FAK in psoriasis and examined the potential impact of FAK inhibitor on psoriasis. A small molecular selective FAK inhibitor, defactinib, was used to evaluate the effect of FAK on psoriasis in in vitro and in vivo functional assays. In our experiments, imiquimod (IMQ)-induced psoriasis mice and human keratinocytes cells were used to study the potential roles and mechanisms of FAK in psoriasis. FAK phosphorylation has been weakly detected in normal intact skin and is markedly elevated upon IMQ treatment. By reducing FAK phosphorylation (p-FAK), defactinib treatment could attenuate psoriasiform inflammation and epidermal hyperplasia in IMQ-treated mice compared with IMQ-induced mice treated with the vehicle. In in vitro studies, resiquimod (R848) increased (p-FAK) and promoted cell proliferation in human keratinocytes cells, while defactinib reversed this effect. Mechanistically, defactinib can alleviate the proliferation via JNK/YB1 pathway in vitro and in vivo. Defactinib significantly attenuates psoriasiform inflammation and epidermal hyperproliferation through the inhibition of the FAK-mediated axis. The downregulation of phosphorylated FAK then suppressed the activation of JNK/YB1 protein signaling pathway in psoriasis. Our work highlights targeting FAK as a potentially effective strategy for the treatment of psoriasis.

Inhibition of protein translational machinery in triple-negative breast cancer as a promising therapeutic strategy.

Y-box binding protein-1 (YB-1) is a proto-oncogenic protein associated with protein translation regulation. It plays a crucial role in the development and progression of triple-negative breast cancer (TNBC). In this study, we describe a promising approach to inhibit YB-1 using SU056, a small-molecule inhibitor. SU056 physically interacts with YB-1 and reduces its expression, which helps to restrain the progression of TNBC. Proteome profiling analysis indicates that the inhibition of YB-1 by SU056 can alter the proteins that regulate protein translation, an essential process for cancer cell growth. Preclinical studies on human cells, mice, and patient-derived xenograft tumor models show the effectiveness of SU056. Moreover, toxicological studies have shown that SU056 treatment and dosing are well tolerated without any adverse effects. Overall, our study provides a strong foundation for the further development of SU056 as a potential treatment option for patients with TNBC by targeting YB-1.

The novel bacteriocin BacYB1 produced by Leuconostoc mesenteroides YB1: From recent analytical characterization to biocontrol Verticillium dahliae and Agrobacterium tumefaciens.

Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases.

Microglia-derived exosomes selective sorted by YB-1 alleviate nerve damage and cognitive outcome in Alzheimer's disease.

BACKGROUND: Neuroinflammation is a characteristic pathological change of Alzheimer's Diseases (AD). Microglia have been reported to participate in inflammatory responses within the central nervous system. However, the mechanism of microglia released exosome (EXO) contribute to communication within AD microenvironment remains obscure. METHODS: The interaction between microglia and AD was investigated in vitro and in vivo. RNA-binding protein immunoprecipitation (RIP) was used to investigate the mechanisms of miR-223 and YB-1. The association between microglia derived exosomal YB-1/miR-223 axis and nerve cell damage were assessed using Western blot, immunofluorescence, RT-PCR, ELISA and wound healing assay. RESULTS: Here, we reported AD model was responsible for the M1-like (pro-inflammatory) polarization of microglia which in turn induced nerve cell damage. While M2-like (anti-inflammatory) microglia could release miR-223-enriched EXO which reduced neuroinflammation and ameliorated nerve damage in AD model in vivo and in vitro. Moreover, YB-1 directly interacted with miR-223 both in cell and EXO, and participated in microglia exosomal miR-223 loading. CONCLUSION: These results indicate that anti-inflammatory microglia-mediated neuroprotection form inflammatory damage involves exporting miR-223 via EXO sorted by YB-1. Consequently, YB-1-mediated microglia exosomal sorting of miR-223 improved the nerve cell damage repair, representing a promising therapeutic target for AD.

Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

YB-1 Targeted by miR-509-3-5p Affects Migration and Invasion of Triple­Negative Breast Cancer by Regulating Cellular Epithelial­Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) process is closely linked to metastasis of breast cancer. This article elucidates the role of Y-box binding protein-1 (YB-1) on the migration and invasion of triple-negative breast cancer (TNBC) cells by regulating EMT, and the related mechanism. The expression data of YB-1 and miR-509-3-5p in TNBC samples and normal samples were downloaded from the GEO database. The proliferation, migration, invasion, and EMT of TNBC cells were detected by CCK-8 assay, colony formation assay, wound-healing assay, transwell assay, and immunoblotting analyses. The targeted binding of YB-1 and miR-509-3-5p was validated by luciferase reporter experiment. A xenograft mouse model was constructed to investigate the influence of the miR-509-3-5p/YB-1 axis on TNBC tumor growth in vivo. YB-1 was overexpressed, while miR-509-3-5p was underexpressed in TNBC tumor tissues and various cell lines. Silencing YB-1 depressed cell viability, proliferation, motility, and EMT in vitro, and miR-509-3-5p upregulation exerted the same effects. YB-1 was targeted by miR-509-3-5p. The suppressive effects on the phenotypes of TNBC cells caused by overexpressed miR-509-3-5p were attenuated by YB-1 upregulation. In addition, miR-509-3-5p overexpression restrained TNBC tumor growth and downregulated the YB-1-mediated EMT process in vivo. YB-1 targeted by miR-509-3-5p affects motility of TNBC cells by regulating cellular EMT.

Vaccines and Dementia: Part I. Non-Specific Immune Boosting with BCG: History, Ligands, and Receptors.

Vaccines such as Bacille Calmette-Guérin (BCG) can apparently defer dementia onset with an efficacy better than all drugs known to date, as initially reported by Gofrit et al. (PLoS One14, e0224433), now confirmed by other studies. Understanding how and why is of immense importance because it could represent a sea-change in how we manage patients with mild cognitive impairment through to dementia. Given that infection and/or inflammation are likely to contribute to the development of dementias such as Alzheimer's disease (Part II of this work), we provide a historical and molecular background to how vaccines, adjuvants, and their component molecules can elicit broad-spectrum protective effects against diverse agents. We review early studies in which poxvirus, herpes virus, and tuberculosis (TB) infections afford cross-protection against unrelated pathogens, a concept known as 'trained immunity'. We then focus on the attenuated TB vaccine, BCG, that was introduced to protect against the causative agent of TB, Mycobacterium tuberculosis. We trace the development of BCG in the 1920 s through to the discovery, by Freund and McDermott in the 1940 s, that extracts of mycobacteria can themselves exert potent immunostimulating (adjuvant) activity; Freund's complete adjuvant based on mycobacteria remains the most potent immunopotentiator reported to date. We then discuss whether the beneficial effects of BCG require long-term persistence of live bacteria, before focusing on the specific mycobacterial molecules, notably muramyl dipeptides, that mediate immunopotentiation, as well as the receptors involved. Part II addresses evidence that immunopotentiation by BCG and other vaccines can protect against dementia development.

Y-Box-Binding Proteins Have a Dual Impact on Cellular Translation.

Y-box-binding proteins (YB proteins) are multifunctional DNA- and RNA-binding proteins that play an important role in the regulation of gene expression. The high homology of their cold shock domains and the similarity between their long, unstructured C-terminal domains suggest that Y-box-binding proteins may have similar functions in a cell. Here, we consider the functional interchangeability of the somatic YB proteins YB-1 and YB-3. RNA-seq and Ribo-seq are used to track changes in the mRNA abundance or mRNA translation in HEK293T cells solely expressing YB-1, YB-3, or neither of them. We show that YB proteins have a dual effect on translation. Although the expression of YB proteins stimulates global translation, YB-1 and YB-3 inhibit the translation of their direct CLIP-identified mRNA targets. The impact of YB-1 and YB-3 on the translation of their mRNA targets is similar, which suggests that they can substitute each other in inhibiting the translation of their mRNA targets in HEK293T cells.

ErbB3 Governs Endothelial Dysfunction in Hypoxia-Induced Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension, characterized by vascular remodeling, currently lacks curative therapeutic options. The dysfunction of pulmonary artery endothelial cells plays a pivotal role in the initiation and progression of pulmonary hypertension (PH). ErbB3 (human epidermal growth factor receptor 3), also recognized as HER3, is a member of the ErbB family of receptor tyrosine kinases. METHODS: Microarray, immunofluorescence, and Western blotting analyses were conducted to investigate the pathological role of ErbB3. Blood samples were collected for biomarker examination from healthy donors or patients with hypoxic PH. The pathological functions of ErbB3 were further validated in rodents subjected to chronic hypoxia- and Sugen-induced PH, with or without adeno-associated virus-mediated ErbB3 overexpression, systemic deletion, or endothelial cell-specific ErbB3 knockdown. Primary human pulmonary artery endothelial cells and pulmonary artery smooth muscle cells were used to elucidate the underlying mechanisms. RESULTS: ErbB3 exhibited significant upregulation in the serum, lungs, distal pulmonary arteries, and pulmonary artery endothelial cells isolated from patients with PH compared with those from healthy donors. ErbB3 overexpression stimulated hypoxia-induced endothelial cell proliferation, exacerbated pulmonary artery remodeling, elevated systolic pressure in the right ventricle, and promoted right ventricular hypertrophy in murine models of PH. Conversely, systemic deletion or endothelial cell-specific knockout of ErbB3 yielded opposite effects. Coimmunoprecipitation and proteomic analysis identified YB-1 (Y-box binding protein 1) as a downstream target of ErbB3. ErbB3 induced nuclear translocation of YB-1 and subsequently promoted hypoxia-inducible factor 1/2α transcription. A positive loop involving ErbB3-periostin-hypoxia-inducible factor 1/2α was identified to mediate the progressive development of this disease. MM-121, a human anti-ErbB3 monoclonal antibody, exhibited both preventive and therapeutic effects against hypoxia-induced PH. CONCLUSIONS: Our study reveals, for the first time, that ErbB3 serves as a novel biomarker and a promising target for the treatment of PH.

Upregulated PARP1 confers breast cancer resistance to CDK4/6 inhibitors via YB-1 phosphorylation.

BACKGROUND: Cyclic-dependent kinase (CDK) 4/6 kinases, as the critical drivers of the cell cycle, are involved in the tumor progression of various malignancies. Pharmacologic inhibitors of CDK4/6 have shown significant clinical prospects in treating hormone receptor-positive and human epidermal growth factor receptor-negative (HR + /HER2-) breast cancer (BC) patients. However, acquired resistance to CDK4/6 inhibitors (CDK4/6i), as a common issue, has developed rapidly. It is of great significance that the identification of novel therapeutic targets facilitates overcoming the CDK4/6i resistance. PARP1, an amplified gene for CDK4/6i-resistant patients, was found to be significantly upregulated during the construction of CDK4/6i-resistant strains. Whether PARP1 drives CDK4/6i resistance in breast cancer is worth further study. METHOD: PARP1 and p-YB-1 protein levels in breast cancer cells and tissues were quantified using Western blot (WB) analysis, immunohistochemical staining (IHC) and immunofluorescence (IF) assays. Bioinformatics analyses of Gene Expression Profiling Interactive Analysis (GEPIA), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets were applied to explore the relationship between YB-1/PARP1 protein levels and CDK4/6i IC50. Cell Counting Kit-8 (CCK-8) and crystal violet staining assays were performed to evaluate cell proliferation rates and drug killing effects. Flow cytometry assays were conducted to assess apoptosis rates and the G1/S ratio in the cell cycle. An EdU proliferation assay was used to detect the DNA replication ratio after treatment with PARP1 and YB-1 inhibitors. A ChIP assay was performed to assess the interaction of the transcription factor YB-1 and associated DNA regions. A double fluorescein reporter gene assay was designed to assess the influence of WT/S102A/S102E YB-1 on the promoter region of PARP1. Subcutaneous implantation models were applied for in vivo tumor growth evaluations. RESULTS: Here, we reported that PARP1 was amplified in breast cancer cells and CDK4/6i-resistant patients, and knockdown or inhibition of PARP1 reversed drug resistance in cell experiments and animal models. In addition, upregulation of transcription factor YB-1 also occurred in CDK4/6i-resistant breast cancer, and YB-1 inhibition can regulate PARP1 expression. p-YB-1 and PARP1 were upregulated when treated with CDK4/6i based on the WB and IF results, and elevated PARP1 and p-YB-1 were almost simultaneously observed during the construction of MCF7AR-resistant strains. Inhibition of YB-1 or PAPR1 can cause decreased DNA replication, G1/S cycle arrest, and increased apoptosis. We initially confirmed that YB-1 can bind to the promoter region of PARP1 through a ChIP assay. Furthermore, we found that YB-1 phosphorylated at S102 was crucial for PARP1 transcription according to the double fluorescein reporter gene assay. The combination therapy of YB-1 inhibitors and CDK4/6i exerted a synergistic antitumor effect in vitro and in vivo. The clinical data suggested that HR + /HER2- patients with low expression of p-YB-1/PARP1 may be sensitive to CDK4/6i in breast cancer. CONCLUSION: These findings indicated that a ''YB-1/PARP1'' loop conferred resistance to CDK4/6 inhibitors. Furthermore, interrupting the loop can enhance tumor killing in the xenograft tumor model, which provides a promising strategy against drug resistance in breast cancer.

Y-box protein-1 modulates circSPECC1 to promote glioma tumorigenesis via miR-615-5p/HIP1/AKT axis.

Y-box binding protein-1 (YB-1) is upregulated in glioma and plays an important role in its occurrence and drug resistance. However, the involved regulatory processes and downstream pathways are still unclear. Since various circular RNAs (circRNAs) and microRNAs (miRNAs) also play roles in the pathogenesis of glioma, we hypothesize that YB-1 may exert its function through a circRNA-miRNA-protein interaction network. In this study, we use the RNA binding protein immunoprecipitation assay and quantitative reverse transcription polymerase chain reaction to determine the circRNAs involved in the regulation of YB-1 and further elucidate their biological functions. The level of circSPECC1 (hsa_circ_0000745) modulated by YB-1 is significantly upregulated in the U251 and U87 glioma cell lines. Downregulation of circSPECC1 markedly inhibits the proliferation and invasiveness of U251 and U87 cells by inducing apoptosis. Bioinformatics analysis reveals that miR-615-5p could interact with circSPECC1 and huntingtin-interacting protein-1 (HIP-1). Then we determine the interactions between miR-615-5p, circSPECC1, and HIP1 using dual luciferase reporter system and pull-down assays. Mechanistic analysis indicates that the downregulation of circSPECC1 results in a decreased HIP1 expression. This study demonstrates that circSPECC1 modulated by YB-1 is increased in glioma cell lines. In addition, circSPECC1 promotes glioma growth through the upregulation of HIP1 by sponging miR-615-5p and targeting the HIP1/AKT pathway. This indicates that YB-1 and circSPECC1 may both be promising targets for glioma treatment.

Targeted IFNγ induction by a genetically engineered Salmonella typhimurium is the key to the liver metastasis inhibition in a mouse model of pancreatic neuroendocrine tumor.

Background: Liver metastasis is one of the primary causes of death for the patients with pancreatic neuroendocrine tumors (PNETs). However, no curative therapy has been developed so far. Methods: The anti-tumor efficacy of a genetically engineered tumor-targeting Salmonella typhimurium YB1 was evaluated on a non-functional INR1G9 liver metastasis model. Differential inflammatory factors were screened by Cytometric Bead Array. Antibody depletion assay and liver-targeted AAV2/8 expression vector were used for functional evaluation of the differential inflammatory factors. Results: We demonstrated that YB1 showed significant anti-tumor efficacy as a monotherapy. Since YB1 cannot infect INR1G9 cells, its anti-tumor effect was possibly due to the modulation of the tumor immune microenvironment. Two inflammatory factors IFNγ and CCL2 were elevated in the liver after YB1 administration, but only IFNγ was found to be responsible for the anti-tumor effect. Liver-targeted expression of IFNγ caused the activation of macrophages and NK cells, and reproduced the therapeutic effect of YB1 on liver metastasis. Conclusion: We demonstrated that YB1 may exhibit anti-tumor effect mainly based on IFNγ induction. Targeted IFNγ therapy can replace YB1 for treating liver metastasis of PNETs.

Knockdown of DLK4 inhibits non-small cell lung cancer tumor growth by downregulating CKS2.

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is considered as the most common type of cancer. DLX4 was originally identified as a ß-globin gene suppressor in red blood cells, which plays critical roles in several types of cancers. However, the role and related mechanism of DLX4 in NSCLC are still unclear. The study aimed to uncover the expression of DLX4 in human NSCLC cells and tissues, reveal its possible role in NSCLC, and investigate the underlying mechanisms. Immunoblot and TCGA database were used to detect the expression of DLX4 in human NSCLC cells and tissues. CCK-8, colony formation, and FCM assays were conducted to detect the effects of DLX4 on the viability and cell cycle of NCI-H2170 and A549 cells. Immunoblot assays were further performed to investigate the possible mechanism underlying DLX4 affecting the growth of NSCLC. We revealed that knockdown of DLX4 inhibited NSCLC cell proliferation. We further revealed that DLX4 knockdown induced the NSCLC cell cycle arrest. Our results further showed that downregulation of DLX4 suppressed YB-1 expression, which further suppressed CKS2 expression, thereby suppressing tumor growth of NSCLC. In conclusion, DLX4 has the potential to serve as a promising drug for NSCLC treatment.

Targeting YB-1 via entinostat enhances cisplatin sensitivity of pleural mesothelioma in vitro and in vivo.

Pleural mesothelioma (PM) is characterized by poor prognosis and limited therapeutic options. Y-box-binding protein 1 (YB-1) was shown to drive growth and migration of PM cells. Here, we evaluated the effect of genetic and pharmacological targeting of YB-1 on PM growth and response to cisplatin and radiation treatment. YB-1 knockdown via siRNA resulted in reduced PM cell growth, which significantly correlated with wt BAP1 and mutant NF2 and P53 status. Entinostat inhibited YB-1 deacetylation and its efficacy correlated with YB-1 knockdown-induced growth inhibition in 20 PM cell lines. Tumor growth inhibition by siRNA as well as entinostat was confirmed in mouse xenotransplant models. Furthermore, both YBX1-targeting siRNA and entinostat enhanced sensitivity to cisplatin and radiation. In particular, entinostat showed strong synergistic interactions with cisplatin which was linked to significantly increased cellular platinum uptake in all investigated cell models. Importantly, in a mouse model, the combination of cisplatin and entinostat also resulted in stronger growth inhibition than each treatment alone. Our study highlights YB-1 as an attractive target in PM and demonstrates that targeting YB-1 via entinostat is a promising approach to enhance cisplatin and radiation sensitivity.

Proteomic analysis of zebrafish folliculogenesis identifies YB-1 (Ybx1/ybx1) as a potential gatekeeping molecule controlling early ovarian folliculogenesis.

As in mammals, ovarian folliculogenesis in teleosts also consists of two phases: the primary growth (PG) and secondary growth (SG) phases, which are analogous to the preantral and antral phases respectively in mammals. In this study, we performed a proteomic analysis on zebrafish follicles undergoing the PG-SG transition aiming to identify factors involved in the event. Numerous proteins showed significant changes, and the most prominent one was Y-box binding protein 1 (YB-1; Ybx1/ybx1), a transcription factor and mRNA-binding protein. YB-1 belongs to the Y-box binding protein family, which also includes the gonad-specific YB-2. Interestingly, phylogenetic analysis showed no YB-2 homolog in zebrafish. Although ybx1 mRNA was expressed in various tissues, its protein Ybx1 was primarily produced in the gonads, similar to YB-2 in other species. In the ovary, Ybx1 protein started to appear in early follicles newly emerged from the germ cell cysts, reached the highest level in late PG oocytes, but decreased precipitously when the follicles entered the SG phase. In PG follicles, Ybx1 might function as a key component of the messenger ribonucleoprotein particles (mRNPs) in association with other RNA-binding proteins. Similar to mammalian YB-1, zebrafish Ybx1 also contains functional signals that determine its intracellular localization. In conclusion, Ybx1 may play dual roles of YB-1 and YB-2 in zebrafish. In the ovary, Ybx1 binds mRNAs to stabilize them while preventing their translation. At PG-SG transition, Ybx1 is removed to release the masked mRNAs for translation into functional proteins, leading to follicle activation.

Histone H1.2 Represses the Transcription of the p16 Tumor Suppressor Gene.

BACKGROUND/AIM: CDK inhibitor p16 plays a pivotal role in the induction of cellular senescence and functions as a tumor suppressor. Here, we demonstrate that histone H1.2 is involved in p16 repression. MATERIALS AND METHODS: Cells were transfected with siRNAs and subjected to quantitative reverse transcription-polymerase chain reaction, immunoblotting and chromatin immunoprecipitation (ChIP) assay. RESULTS: The decrease in H1.2 by oncogenic RAS was associated with increased levels of p16. Depletion of H1.2 selectively increased p16, but not alternative reading frame (ARF) mRNA. ChIP assay showed that H1.2 directly bound to the p16 promoter. Interestingly, silencing YB-1, a component of H1.2 complex, decreased the expression levels of H1.2, resulting in decreased binding of H1.2 on the p16 promoter. CONCLUSION: These results provide a model in which H1.2 is positively regulated by YB-1 and directly binds to and represses the transcription of p16.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

TabHLH95-TaNF-YB1 module promotes grain starch synthesis in bread wheat.

Starch is the most abundant substance in wheat (Triticum aestivum L.) endosperm and provides the major carbohydrate energy for human daily life. Starch synthesis-related (SSR) genes are believed to be spatiotemporally specific, but their transcriptional regulation remains unclear in wheat. Here, we investigate the role of the basic helix-loop-helix (bHLH) transcription factor TabHLH95 in starch synthesis. TabHLH95 is preferentially expressed in the developing grains in wheat and encodes a nucleus localized protein without autoactivation activity. The Tabhlh95 knockout mutants display smaller grain size and less starch content than wild type, whereas overexpression of TabHLH95 enhances starch accumulation and significantly improves thousand grain weight. Transcriptome analysis reveals that the expression of multiple SSR genes is significantly reduced in the Tabhlh95 mutants. TabHLH95 binds to the promoters of ADP-glucose pyrophosphorylase large subunit 1 (AGPL1-1D/-1B), AGPL2-5D, and isoamylase (ISA1-7D) and enhances their transcription. Intriguingly, TabHLH95 interacts with the nuclear factor Y (NF-Y) family transcription factor TaNF-YB1, thereby synergistically regulating starch synthesis. These results suggest that the TabHLH95-TaNF-YB1 complex positively modulates starch synthesis and grain weight by regulating the expression of a subset of SSR genes, thus providing a good potential approach for genetic improvement of grain productivity in wheat.

YB1 participated in regulating mitochondrial activity through RNA replacement.

As a relic of ancient bacterial endosymbionts, mitochondria play a central role in cell metabolism, apoptosis, autophagy, and other processes. However, the function of mitochondria-derived nucleic acids in cellular signal transduction has not been fully elucidated. Here, our work has found that Y-box binding protein 1 (YB1) maintained cellular autophagy at a moderate level to inhibit mitochondrial oxidative phosphorylation. In addition, mitochondrial RNA was leaked into cytosol under starvation, accompanied by YB1 mitochondrial relocation, resulting in YB1-bound RNA replacement. The mRNAs encoded by oxidative phosphorylation (OXPHOS)-associated genes and oncogene HMGA1 (high-mobility group AT-hook 1) were competitively replaced by mitochondria-derived tRNAs. The increase of free OXPHOS mRNAs released from the YB1 complex enhanced mitochondrial activity through facilitating translation, but the stability of HMGA1 mRNA was impaired without the protection of YB1, both contributing to breast cancer cell apoptosis and reactive oxygen species production. Our finding not only provided a new potential target for breast cancer therapy but also shed new light on understanding the global landscape of cellular interactions between RNA-binding proteins and different RNA species.

Promotion of liver fibrosis by Y-box binding protein 1 via the attenuation of transforming growth factor-beta 3 transcription.

Background: Spurred by the seriousness of liver fibrosis, we evaluated the correlation between Y-box binding protein 1 (YB-1) and transforming growth factor-beta 3 (TGF-ß3) expression levels in the signaling pathways of the disease. Methods: Based on a mouse model of carbon tetrachloride-induced liver fibrosis, YB-1 overexpression lentivirus was used to explore the effect of YB-1 on liver fibrosis in vivo. In addition, a hepatic stellate cell (HSC) activation model in the HSC line LX-2 was developed using TGF-ß1. Western blot assays were used to investigate the effects of YB-1 overexpression and knockdown on liver fibrosis. Finally, chromatin immunoprecipitation and luciferase reporter assays were used to elucidate the relationship between YB-1 and its downstream signaling pathways. Results: YB-1 was overexpressed in fibrotic liver tissue, which enhanced both fibrosis and the relative protein expressions of the TGF-ß pathway. Moreover, YB-1 overexpression promoted HSC activation in response to TGF-ß1 stimulation, but its knockdown inhibited liver fibrosis in vitro. Both in vitro and in vivo experiments indicated the expression of TGF-ß3 in the YB-1 overexpression group to be suppressed, and liver fibrosis was more obvious in the YB-1-overexpression group than in the YB-1-inhibition group. YB-1 attenuated TGF-ß3 transcription by binding to its promoter, which is involved in the effect of YB-1 on liver fibrosis. Conclusions: YB-1 overexpression in HSCs promoted liver fibrosis by attenuating TGF-ß3 transcription.